Project Summary Phase Transition-Mediated Tau Function and Dysfunction The proposed research will decipher the molecular mechanism of liquid-liquid phase separation (LLPS)-mediated Tau function and dysfunction. Phase-separated Tau droplets enrich tubulin and facilitate microtubule assembly (Tau function), whereas persistent droplets lead to protein aggregation. Utilizing prior extensive experience with disordered protein systems, the investigators will determine how LLPS links Tau loss-of-function and gain-of-toxic dysfunction in three stages: They will characterize how pathologic post-translational modifications (PTMs; hyperphosphorylation and hyperacetylation) modulate Tau LLPS-mediated microtubule assembly and protein aggregation (Aim 1); they will track Tau conformations that are key to both LLPS-mediated Tau function and dysfunction (Aim 2); and, they will determine how co- aggregating proteins initiate/ facilitate/ synergize Tau aggregation (Aim 3). The first Aim will utilize ensemble spectroscopy and time-lapse microscopy techniques to understand the role of PTMs in LLPS-mediated function and dysfunction. Phase transition maps of different Tau variants will be generated to characterize how different PTMs alter Tau LLPS. Effects of Tau variant co-partitioning in droplets or fibrillar aggregates on Tau function and dysfunction will also be studied. The second Aim will utilize ultrasensitive fluorescence spectroscopy techniques to track conformational conversions as monomeric Tau forms condensed droplets and as liquid droplets transform to solid states, i.e., functional (in microtubule assembled filaments) and/or dysfunctional states (amyloid fibril states). The third Aim will determine the role of co-aggregating proteins in Tau LLPS and subsequent protein aggregation. Synergistic interaction between co-phase separating proteins will be characterized. Mechanism of aggregate cross-seeding will provide insights into protein co-misfolding.